1. Field of the Invention
The present invention relates to the field of nucleic acid detection and/or quantification. More particularly, the present invention concerns novel approaches to detection and/or quantification of gene expression, single nucleotide polymorphisms (SNPs), protein-protein interaction, real time PCR and/or pathogen typing.
2. Description of Related Art
Methods of precise and highly sensitive detection and/or quantification of nucleic acids are of use for a variety of medical, forensic, epidemiological, public health, biological warfare and other applications. A variety of molecular biology and genomic techniques would benefit from the availability of precise and sensitive methods for nucleic acid detection and/or quantification.
DNA microarrays provide a platform for exploring the genome, including analysis of gene expression by hybridization with sequence specific oligonucleotide probes attached to chips in precise arrays. (E.g., Schena et al., Science 270:467–470, 1995; Shalon et al., Genome Res. 6:639–645, 1996; Pease et al., Proc. Natl. Acad. Sci. USA 91:5022–26, 1994). Microarray technology is an extension of previous hybridization-based methods, such as Southern and Northern blotting, that have been used to identify and quantify nucleic acids in biological samples (Southern, J. Mol. Biol. 98:503–17, 1975; Pease et al., Proc. Natl. Acad. Sci. USA 93:10614–19, 1996). Identification of a target nucleic acid in a sample typically involves fluorescent detection of the nucleic acid hybridized to an oligonucleotide at a particular location on the array. Fluorescent detection is too insensitive to detect very low levels of a target nucleic acid in a sample. It is also more qualitative than quantitative. Thus, detection of small changes in the level of expression of a particular gene, as might be attempted for high through-put screening of potential inhibitors and/or activators of gene expression, may not be feasible using a fluorescence detection system with microarrays. More accurate and sensitive methods for nucleic acid quantification are needed.
Real time PCR™ (polymerase chain reaction) is another technique for which accurate and sensitive quantification are needed (e.g., Model 770 TaqMan® system, Applied Biosystems, Foster City, Calif.). Typically, if the target of interest is present, it will be amplified by replication using flanking primers and a nucleic acid polymerase. A probe, which may consist of a complementary oligonucleotide with attached reporter and quencher dyes, is designed to bind to the amplified target nucleic acid between the two primer-binding sites. The nuclease activity of the polymerase cleaves the probe, resulting in an increase in fluorescence of the reporter dye after it is separated from the quencher. PCR based fluorescence detection of target nucleic acids is more sensitive, due to the amplification effect of the technique. However, precise quantification of the amount of target present may be complicated by a variety of factors, such as contaminating nuclease activity or variability in the efficiency of amplification.
Single nucleotide polymorphisms (SNPs) are of increasing interest in molecular biology, genomics and disease diagnostics. SNP detection may be used for haplotype construction in genetic studies to identify and/or detect genes associated with various disease states, as well as drug sensitivity or resistance. SNPs may be detected by a variety of techniques, such as DNA sequencing, fluorescence detection, mass spectrometry or DNA microarray hybridization (e.g., U.S. Pat. Nos. 5,885,775; 6,368,799). Existing methods of SNP detection may suffer from insufficient sensitivity or an unacceptably high level of false positive and/or false negative results. A need exists for more sensitive and accurate methods of detecting SNPs.
Pyrophosphate based detection systems have been used for DNA sequencing (e.g., Nyren and Lundin, Anal. Biochem. 151:504–509, 1985; U.S. Pat. Nos. 4,971,903; 6,210,891; 6,258,568; 6,274,320, each incorporated herein by reference). The method uses a coupled reaction wherein pyrophosphate is generated by an enzyme-catalyzed process, such as nucleic acid polymerization. The pyrophosphate is used to produce ATP, in an ATP sulfurylase catalyzed reaction with adenosine 5′-phosphosulphate (APS). The ATP in turn is used for the production of light in a luciferin-luciferase coupled reaction. The present invention provides a novel method of pyrophosphate-based detection for use in SNP detection, gene expression assays, protein-protein interaction, real time PCR, pathogen typing and other applications.